Capillary electrophoretic apparatus

ABSTRACT

A sample is separated and separated components thereof are successively fed to a part to be detected. A laser beam of at least 600 nm from a laser beam source of an optical measuring part is applied to the part to be detected through a dichroic mirror and a lens, for making a fluorochrome bonded to the separated components absorb multiphotons, exciting the fluorochrome and making the same fluoresce. The optical measuring part captures the fluorescence so that photomultipliers detect fluorescence of not more than 510 nm in wavelength, fluorescence longer than 510 nm and not more than 560 nm in wavelength, fluorescence longer than 560 nm and not more than 580 nm in wavelength and fluorescence longer than 580 in wavelength respectively. Thus, a capillary electrophoretic apparatus can detect fluorescence from a fluorochrome bonded to samples as a label without influence by Raman scattering or Rayleigh scattering.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a capillary electrophoreticapparatus for separating and analyzing a biopolymer such as protein ornucleic acid.

[0003] Such a capillary electrophoretic apparatus is applied to sequencedetermination for DNA. The capillary electrophoretic apparatus for DNAsequence determination electrophoreses a DNA fragment sample prepared bylabeling a primer or a terminator with a fluorochrome and detectsfluorescence from the DNA fragment sample during electrophoresis fordetermining the base sequence.

[0004] 2. Description of the Prior Art

[0005] A DNA sequencer having high sensitivity, a high speed and highthroughput is necessary for sequence determination for DNA such as ahuman genome having long base sequence. As an example, capillaryelectrophoresis employing a capillary column charged with a gel in placeof slab gel electrophoresis employing a flat plate type slab gel isproposed. With such a capillary column, a sample can not only be readilyhandled or injected but also electrophoresed at a high speed anddetected in high sensitivity as compared with the slab gel. If a highvoltage is applied to the slab gel, a band is spread or a temperaturegradient is caused due to influence by Joulean heat. However, thecapillary column hardly causes such a problem and can perform detectionin high sensitivity with small band spreading even if performinghigh-speed electrophoresis with application of a high voltage.

[0006] A multi-capillary electrophoretic apparatus prepared by arranginga plurality of capillary columns is also proposed.

[0007] An automatic DNA sequencer utilizes a fluorochrome foridentifying the four types of bases forming DNA. A Rhodamine derivativesuch as R6G, R-110 or ROX or a fluorescein derivative such as FAM isutilized as the fluorochrome. An argon ion laser unit having dominantwavelengths of 488.0 nm and 514.5 nm is utilized as a laser beam source.

[0008] However, both of the wavelengths of 488.0 nm and 514.5 nm areseparate from the absorption maximum wavelengths of the fluoresceinderivatives and the Rhodamine derivatives. While fluorescein derivativeshaving an absorption maximum wavelength of 493.5 nm are not much reducedin efficiency, the Rhodamine derivatives, which are excited at 514.5 nmalthough its absorption maximum wavelength is 550 nm, has inferiorefficiency.

[0009] In order to solve this problem, there has been made an attempt(referred to as an energy transfer method) of introducing both of thefluorescein derivative and the Rhodamine derivative into the samemolecule when using the Rhodamine derivative as a label therebyimproving the efficiency of the Rhodamine derivative through theprinciple of energy transfer.

[0010] The energy transfer method is superior to general methods, butyet has the following problems:

[0011] 1. It is technically difficult to introduce a plurality offluorochromes into the same molecule, and the cost is increasedfollowing this difficulty.

[0012] 2. Extreme influence is exerted by Raman scattering since theexcitation wavelength is in the visible region. When excited at 488 nm,a Raman scattering line of water around 516 nm forms background noise ofa channel detecting the fluorescein derivative having a fluorescencemaximum at 510 nm to reduce an S-N (signal-to-noise) ratio.

[0013] 3. Influence is exerted by Rayleigh scattering to readily reducethe S-N ratio.

[0014] Considering a multi-capillary electrophoretic apparatus in whicha plurality of capillary columns are so arranged that a plurality ofsamples are injected into the capillary columns and simultaneouslyelectrophoresed in all capillary columns, one ends of the plurality ofcapillary columns defining a sample injection side are two-dimensionallyarranged and fixed by a sample injection side holder while the otherends defining a detection side are aligned with each other on a planeand fixed by a detection side holder for forming a capillary array. Thedetection side holder is provided with a slot along the arrangement ofthe capillary columns, and parts of the capillary columns exposedthrough the slot define a part to be detected. When separating anddetecting a sample containing a DNA fragment labeled in four types witha fluorescent material, excitation light is applied to the part to bedetected for detecting fluorescence generated from sample componentselectrophoresed to the part to be detected thereby identifying thesample components.

[0015] The prior art employs an epi-optical system comprising acondenser lens condensing and projecting excitation light onto eachcapillary column on the part to be detected and receiving thefluorescence generated from the sample electrophoresed in the capillarycolumn as an objective lens for projecting the excitation light andreceiving the fluorescence through the same lens as an excitation-lightreceiving optical system. The objective lens is scanned along a straightline parallel to the plane of arrangement of the capillary columns onthe part to be detected and perpendicular to the electrophoresisdirection, thereby detecting the fluorescence as to all capillarycolumns.

[0016] In such an optical system, the objective lens is preferablyarranged in proximity to the part to be detected for collecting themaximum amount of fluorescence in consideration of detectionsensitivity. Therefore, the condenser lens having a short focal lengthis employed as the objective lens.

[0017] When employing the condenser lens having a short focal length asthe objective lens, the amount of collected fluorescence is reduced toreduce the detection sensitivity if the position of the part to bedetected of the capillary array slightly deviates in the direction ofapplication of the excitation light. Therefore, high working accuracy isrequired when preparing the detection side holder and fixing thecapillary columns to the detection side holder.

[0018] In the multi-capillary electrophoretic apparatus, the capillarycolumns are fixed to cassette holders on a sample introduction side andthe detection side. The cassette holders two-dimensionally arrange thecapillary columns on the sample introduction side and planarly align thesame with each other on the detection side.

[0019] When charging each capillary column with a polymer, one end ofthe capillary column is stuck into and fixed to an elastic member suchas a rubber stopper or fixed to a dedicated holder for polymer chargingwith an adhesive for filling up a clearance. The polymer is charged intothe capillary column by fixing the elastic member or the dedicatedholder to a vessel storing the polymer so that the end of the capillarycolumn is dipped in the polymer, sealing the vessel and pressurizing thevessel with a pump for press-filling the polymer into the capillarycolumn, or by connecting the elastic member or the dedicated holder to apump, dipping another end of the capillary column into the polymer anddecompressing the capillary column with the pump for inhaling thepolymer into the capillary column.

[0020] When charging the polymer into the capillary column bypress-filling or inhaling in the method of sticking and fixing thecapillary column into and to the elastic member, pressure resistance ofthe elastic member may be so insufficient that the polymer cannot besmoothly charged into the capillary column. On the other hand, in themethod of fixing the capillary column to the dedicated holder forpolymer charging with an adhesive, it may be impossible to smoothlycharge the polymer into the capillary column due to insufficient supplyof the adhesive, to result in an inferior manufacturing yield.

SUMMARY OF THE INVENTION

[0021] A first objective of the present invention is to performefficient detection in a capillary electrophoretic apparatus.

[0022] A second objective of the present invention is to provide acapillary cassette capable of reliably charging all capillary columnswith polymers with a high yield in a multi-capillary electrophoreticapparatus.

[0023] A first aspect of the present invention for performing efficientdetection comprises detection means exciting a fluorochrome bonded to asample component as a label for making the same fluoresce and detectingthe generated fluorescence without influence by Raman scattering orRayleigh scattering. In a capillary electrophoretic apparatus accordingto the present invention, the detection means applies excitation lighthaving a wavelength longer than the fluorescent wavelength of thefluorochrome, excites the fluorochrome by multiphoton absorption anddetects fluorescence generated from the fluorochrome. In other words,this aspect utilizes a multiphoton absorption method of applying light(the excitation light having a longer wavelength than the fluorescentwavelength of the fluorochrome) having energy of one photon smaller thanexcitation energy for the fluorochrome to the sample bonded with thefluorochrome and making the fluorochrome absorb multiphotons therebyexciting the fluorochrome and making the same fluoresce.

[0024] In the multiphoton absorption method, both a fluoresceinderivative and a Rhodamine derivative can be excited with a common laserwavelength. Therefore, it is not necessary to introduce a plurality offluorochromes into the same molecule.

[0025] The excitation wavelength used in the multiphoton absorptionmethod may be set in a range from 400 nm to 2 μm, and preferably is setin the near infrared region of at least 600 nm. With the excitationwavelength of at least 600 nm, most of the Raman scattering lineoutgoing from the wavelength is Stokes Raman scattered light having awavelength of at least 600 nm. Therefore, the Raman scattering lightdoes not form background noise in detection of fluorescence fromfluorescein or Rhodamine.

[0026] Furthermore, the intensity of Rayleigh scattering is in inverseproportion to the sixth power of the wavelength, and hence theexcitation light of a longer wavelength region exceeding 600 nm utilizedin the multiphoton absorption method is superior to an argon laser beamfor suppressing Rayleigh scattering.

[0027] Thus, the fluorescence from the fluorochrome can be detected in ahigh S-N ratio while suppressing influence by Raman scattering andRayleigh scattering by comprising the detection means employing themultiphoton absorption method, applying the light of a wavelength longerthan the fluorescent wavelength of the fluorochrome to the fluorochromefor exciting the same and detecting the fluorescence thereof.

[0028] A second aspect of the present invention for performing efficientdetection is to relieve requirement for working accuracy at the time ofpreparing a detection side holder and fixation of capillary columns tothe detection holder, fix the position of a part to be detected of acapillary array to the apparatus with excellent reproducibility, andsuppress reduction of detection sensitivity. in a multi-capillaryelectrophoretic apparatus The multi-capillary electrophoretic apparatusto which this aspect is applied comprises a capillary array in which aplurality of capillary columns are so arranged that one ends defining asample injection side are fixed by a sample injection side holder, theother ends defining a detection side are aligned with each other on aplane and fixed by a detection side holder and a part to be detected isprovided on the position of the detection side holder, a multi-capillaryarray electrophoresis part to which the sample injection side holder andthe detection side holder are fixed so that samples are injected intothe capillary columns, the ends on the sample injection side are dippedinto a buffer solution, the ends on the detection side are dipped intoanother buffer solution and an electrophoresis voltage is appliedthrough both buffer solutions for performing electrophoresis in allcapillary columns, and a detection part applying light to the part to bedetected of the capillary array and detecting light affected byinteraction with the samples. According to this aspect, themulti-capillary array electrophoresis part includes a detection sideholder fixing part fixing the detection side holder and a parallelismadjusting mechanism adjusting the parallelism between the detection partand the part to be detected.

[0029] Detection can be performed in constant sensitivity regardless ofthe position of the part to be detected by adjusting the parallelismbetween the detection part and the part to be detected by theparallelism adjusting mechanism.

[0030] The detection system of the detection part may be either ascanning system of sequentially detecting the capillary columns one byone on the part to be detected or an image system of collectivelycapturing the capillary columns on the part to be detected as an image.

[0031] The detection part in the scanning system comprises anepi-optical system condensing and projecting light onto one of thecapillary columns on the part to be detected while receiving lightaffected by interaction with the samples and a scanning mechanismreciprocally moving the epi-optical system along a straight lineparallel to the plane of arrangement on the part to be detected of thecapillary array and perpendicular to the electrophoresis direction, andthe parallelism adjusting mechanism adjusts the parallelism between ascanning axis of the epi-optical system and the part to be detected inthis case.

[0032] In the parallelism adjustment, the scanning system fixes thedetection side holder to the detection side holder fixing part,thereafter drives the scanning mechanism to reciprocate the epi-opticalsystem in the direction perpendicular to the electrophoresis direction,and adjusts the parallelism between the scanning axis of the epi-opticalsystem and the part to be detected by the parallelism adjustingmechanism on the basis of a current detection signal of the detectionpart.

[0033] The image system can be provided with an imaging optical systemand a line sensor described in, for example, U.S. Pat. No. 5,534,703. Inthis case, the parallelism adjusting mechanism may adjust an opticalaxis of the imaging optical system.

[0034] A mode of the parallelism adjusting mechanism is preferably agate adjusting mechanism adjusting a mounting angle of the detectionside holder fixing part by rotation of a screw. Consequently, theparallelism between the scanning axis of the epi-optical system and thepart to be detected can be adjusted in a simple structure through asimple operation.

[0035] Another mode of the parallelism adjusting mechanism preferablycomprises an actuator automatically adjusting a gate angle of thedetection side holder fixing part in correspondence to the detectionsignal at the time of scanning the epi-optical system. Consequently, aburden on an operator can be reduced.

[0036] Furthermore, the detection part preferably comprises anepi-optical system condensing and projecting light onto each capillarycolumn on the part to be detected and receiving light affected byinteraction with the samples, and a scanning mechanism reciprocallymoving the epi-optical system along a straight line perpendicular to theelectrophoresis direction while automatically adjusting the distancebetween the part to be detected and the epi-optical system incorrespondence to a detection signal at the time scanning the same alongthe straight line. Consequently, the distance between the part to bedetected and the epi-optical system can be rendered suitable withoutproviding a parallelism adjusting mechanism.

[0037] The detection side holder fixing part preferably comprises adetection position member arranged between the part to be detected andthe epi-optical system, having an opening on a position corresponding tothe part to be detected and having a plane in contact with one surfaceof the part to be detected and a detected part pressing member having aplane pressing the part to be detected against the detection positionmember from a side opposite from the detection position member.Consequently, the plurality of capillary columns of the part to bedetected can be fixed onto the plane of the detection position memberwith exceptional reproducibility.

[0038] The inventive multi-capillary electrophoretic apparatus accordingto this aspect fixes the part to be detected of the capillary array ontoa plane of a movable plate by the detected part pressing member andthereafter adjusts a gate angle of the movable plate by a gate adjustingmechanism so that the parallelism between the part to be detected andthe scanning axis of the epi-optical system can be adjusted, wherebyrequirement for working accuracy in preparation of the detection sideholder and fixation of the capillary columns to the detection sideholder can be relaxed, the position of the part to be detected can befixed to the apparatus with excellent reproducibility, and reduction ofdetection sensitivity can be suppressed.

[0039] A capillary cassette according to the present invention capableof reliably charging all capillary columns with polymers in an excellentyield is a capillary cassette in which a plurality of capillary columnsused in a multi-capillary electrophoretic apparatus are bundled so thatfirst ends thereof are cylindrically bundled by a sleeve and clearancesbetween the sleeve and the capillary columns and between the capillarycolumns are sealed with a filler.

[0040] The one ends of the capillary columns cylindrically bundled bythe sleeve have a cylindrical outer shape and hence can be readilymounted on a polymer charger in an airtight manner, whereby thecapillary columns can be readily charged with the polymers through ahigh pressure.

[0041] The sleeve is preferably prepared by shrinking a shrinkablemember, and is preferably a heat-shrinkable tube.

[0042] The capillary column ends can be most densely and cylindricallybundled by passing the one ends of the plurality of capillary columnsthrough the sleeve formed by a shrinkable member and thereaftershrinking the sleeve. When previously applying the filler to thecapillary column surfaces on positions corresponding to the shrinkablemember, the clearances between the capillary columns can be filled upwith the filler without failure in the process of bundling the capillarycolumns. When employing a heat-shrinkable tube as the sleeve, thecapillary column ends can be bundled by simply heating the same with adryer or the like.

[0043] A mounting member for mounting the sleeve on the polymer chargerin an airtight manner is preferably mounted on the sleeve.

[0044] The capillary column ends cylindrically bundled by the sleeve canbe handled similarly to, for example, a pipe of a liquid chromatograph.For example, if a mounting member such as a ferrule, is mounted on thesleeve when charging the capillary columns with the polymers, thecapillary column ends can be fixed to the polymer charger in an airtightmanner.

[0045] It is preferable that a part to be detected in which thecapillary columns are aligned with each other is provided on the side ofthe cylindrically bundled ends while the capillary columns aretwo-dimensionally arranged to define a sample injection part on the sideof the other ends.

[0046] It is possible to dip the capillary column ends in various samplesolutions respectively in sample injection for simultaneously injectingsamples into the respective capillary columns by two-dimensionallyarranging the capillary column ends opposite from the cylindricallybundled ends. Thus, the capillary columns can be reliably charged withthe polymers in an exceptional yield. Furthermore, it is possible toapply a electrophoresis voltage across all capillary columns aftersample injection for simultaneously separating and detecting the samplesin the respective capillary columns.

[0047] The foregoing and other objects, features, aspects and advantagesof the present invention will become more apparent from the followingdetailed description of the present invention when taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1 is a schematic perspective view showing one embodimentapplying the present invention to a multi-capillary electrophoreticapparatus;

[0049]FIG. 2 is a conceptual diagram showing one embodiment of detectionmeans of the embodiment;

[0050]FIG. 3 is a side sectional view schematically showing anotherembodiment;

[0051]FIG. 4 is a front sectional view showing an exemplary capillaryarray mounted on the embodiment;

[0052]FIG. 5 is a left side elevational view of the capillary array;

[0053]FIG. 6 is a top plan view of the capillary array;

[0054]FIG. 7A is a schematic side sectional view showing an opticalsystem in the embodiment,

[0055]FIG. 7B is a schematic perspective view showing a lens panelemployed in FIG. 7A, and

[0056]FIG. 7C is a schematic perspective view showing a filter panelemployed in FIG. 7A;

[0057]FIG. 8A is a schematic sectional view of one embodiment of adetection side holder fixing part and its periphery in the embodiment asviewed from above,

[0058]FIG. 8B is a schematic sectional view taken along the line B-B′ inFIG. 8A and

[0059]FIG. 8C is a schematic sectional view taken along the line C-C′ inFIG. 8A, while FIG. 8A is taken along the lines A-A′ in FIGS. 8B and 8C;

[0060]FIGS. 9A to 9C are a schematic top plan view, a schematic frontelevational view and a schematic right side sectional view showing amovable plate in the detection side holder fixing part respectively;

[0061]FIG. 10 is a perspective view of a capillary cassette of oneembodiment in which a plurality of capillary columns are arranged;

[0062]FIG. 11 is a model diagram showing a procedure of bundlingcapillary column ends with a heat-shrinkable tube;

[0063]FIGS. 12A and 12B are sectional views taken along the lines A-A′and B-B′ showing sections of detection side capillary column ends of theembodiment;

[0064]FIG. 13 is a schematic sectional view showing a state of fixing amounting member for a polymer charger to the capillary column ends; and

[0065]FIG. 14 is a schematic perspective view showing one embodiment ofa multi-capillary electrophoretic apparatus to which the embodiment isapplied.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066]FIG. 1 is a schematic perspective view showing one embodimentapplying the first aspect of the present invention to a multi-capillaryelectrophoretic apparatus, which comprises detection means exciting afluorochrome bonded to a sample component as a label for making the samefluoresce and detecting the fluorescence without influence by Ramanscattering or Rayleigh scattering. FIG. 2 is a conceptual diagramshowing one embodiment of the detection means of this embodiment Thisfigure shows application to a four-color label DNA sequencer.

[0067] A pair of reservoirs 110 and 120 store electrophoresis buffersolutions 112 and 122 respectively, while electrodes 130 and 132 areprovided in the buffer solutions 112 and 122 respectively.

[0068] Respective wells 102 of a sample plate 100 store samples bondedwith fluorochromes having different fluorescent wavelengths such as afluorescein derivative and a Rhodamine derivative in correspondence tothe four types of end bases of DNA fragments. The sample plate 100 isformed with a wiring pattern, in which electrodes are arranged in thewells 102 respectively and connected to a high-voltage wiring cablethrough a connector part 106 respectively.

[0069] A high-voltage switching part 136 switchably connects thereservoir 110 and the sample plate 100 so that wiring can be switched,and an electrophoretic high-voltage power source 134 is connectedbetween the high-voltage switching part 136 and the electrode 132provided in the other reservoir 120 for switching and applying voltagesfor sample injection and electrophoresis.

[0070] In sample injection, one ends 2 a of capillary columns forming acapillary array 2 are inserted one by one into the wells 102 of thesample plate 100, and after the sample injection, the ends 2 a areswitched to the reservoir 110 to be dipped in the buffer solution 112.The other ends 2 b of the capillary columns are dipped in the buffersolution 122 of the other reservoir 120. The second ends 2 b areprovided with a part to be detected 2 c irradiated with excitation lightfrom an optical measuring part 210 detecting the samples by fluorescenceso that the fluorescence is measured.

[0071] The capillary column ends 2 a of the capillary array 2 havetwo-dimensional arrangement corresponding to the arrangement of thewells 102 of the sample plate 100, while the capillary columns arealigned with each other on the part to be detected 2 c and irradiatedwith the excitation light from a direction perpendicular to the plane ofthe arrangement of the capillary columns.

[0072] A moving mechanism (not shown in FIG. 1) switches and arrangesthe sample plate 100 and the reservoir 110 as indicated by the broadarrow so that either one selectively comes into contact with the firstends 2 a of the capillary columns.

[0073] The optical measuring part 210 comprises, for example, a laserbeam source 212 such as a mode-locked titanium sapphire laser unithaving a repetition rate of 78 MHz, a pulse width of 120 to 150 fs, anoscillation wavelength of 700 to 900 nm and an average output of about 1W. The energy of one photon of its laser beam is smaller than excitationenergy for the fluorochromes.

[0074] A dichroic mirror 214 reflecting laser beam is provided on theoptical path of the laser beam from the laser beam source 212, so thatthe laser beam is reflected by the mirror 214 and applied to the part tobe detected 2 c of the capillary array 2 through a lens 216.

[0075] Light from the part to be detected 2 c is transmitted to thedichroic mirror 214 through the lens 216. Fluorescence included in thelight is transmitted through the dichroic mirror 214 and thereafterseparated into prescribed wavelength regions by dichroic mirrors 218,222 and 226. The dichroic mirror 214 transmits light having a shorterwavelength than the laser wavelength of the excitation light in thelight from the lens 216. The light transmitted through the dichroicmirror 214 is transmitted to the dichroic mirror 218 so that lighthaving a wavelength of not more than 510 nm is transmitted through thedichroic mirror 18 and incident upon and detected by a photomultipliertube (PMT) 220, while light having a wavelength longer than 510 nm isreflected by the dichroic mirror 218 and transmitted to the dichroicmirror 222. In the light from the dichroic mirror 218, light having awavelength of not more than 560 nm is reflected by the dichroic mirror222 and incident upon and detected by a photomultiplier tube 224, whilelight having a wavelength longer than 560 nm is transmitted through thedichroic mirror 222 and transmitted to the dichroic mirror 226. In thelight transmitted through the dichroic mirror 222, light having awavelength of not more than 580 nm is reflected by the dichroic mirror226 and incident upon and detected by a photomultiplier tube 228, whilelight having a wavelength longer than 580 nm is transmitted through thedichroic mirror 226 and incident upon and detected by a photomultipliertube 230.

[0076] The optical measuring part 210 is scanned so that the excitationlight reciprocates horizontally across the plane of arrangement of thecapillary columns on the part to be detected 2 c for successivelydetecting all capillary columns. However, illustration of a scanningmechanism is omitted.

[0077] Operations of this embodiment shall now be described withreference to FIGS. 1 and 2.

[0078] In sample injection, the one ends 2 a of the capillary columnsare dipped one by one in the wells 102, while the other ends 2 b of thecapillary columns are collectively dipped in the buffer solution 122 ofthe reservoir 120. The high-voltage switching part 136 is connected tothe sample plate 100, and the electrophoretic high-voltage power source134 applies a high voltage between the wells 102 and the reservoir 120.The samples in the wells 102 are injected into the capillary columns.

[0079] After the sample injection, application of the high voltage istemporarily stopped and the moving mechanism moves the sample plate 100and the reservoir 110, thereby dipping the one ends 2 a of the capillarycolumns on a sample side into the buffer solution 112 of the reservoir110. Thereafter a high voltage is applied between the reservoirs 110 and120 for performing electrophoretic separation. The voltage for sampleinjection into the capillary columns and a electrophoresis power supplyvoltage are, for example, 30 kV and a current capacity is 10 to 30 mA.

[0080] Separated sample components successively pass through the part tobe detected 2 c, and are detected by the optical measuring part 210 atthis time.

[0081] The laser beam from the laser beam source 212 is applied to thepart to be detected 2 c through the dichroic mirror 214 and the lens 216so that the fluorochromes bonded to the samples absorb multiphotons andare excited to fluoresce. The optical measuring part 210 captures thefluorescence so that the photomultiplier tubes 220, 224 and 228 detectfluorescence having a wavelength of not more than 510 nm, fluorescencehaving a wavelength longer than 510 nm and not more than 560 nm,fluorescence having a wavelength longer than 560 nm and not more than580 nm and fluorescence having a wavelength longer than 580respectively.

[0082] Base sequence can be determined by bonding the fluorescencehaving a wavelength of not more than 510 nm, the fluorescence having awavelength longer than 510 nm and not more than 560 nm, fluorescencehaving a wavelength longer than 560 nm and not more than 580 nm and thefluorescence having a wavelength longer than 580 to DNA fragment samplesfor the respective bases respectively.

[0083] Since a Raman scattering line generated in the part to bedetected 2 c due to irradiation with the laser beam has a wavelength atleast 700 nm, it does not form background noise in fluorescencedetection. Furthermore, the intensity of Rayleigh scattering isadvantageously smaller than that of a conventional argon laser beam. Inaddition, four types of fluorochromes can be efficiently excited with asingle laser wavelength due to the multiphoton absorption method,whereby it is not necessary to introduce a plurality of fluorochromesinto the same molecule.

[0084] The optical measuring part 210 is not restricted to thisembodiment but may have any system so far as the same can excitefluorochromes bonded to samples by multiphoton absorption for making thesame fluoresce and detect the fluorescence.

[0085] While the present invention is applied to a multi-capillaryelectrophoretic apparatus in this embodiment, the present invention isalso applicable to electrophoresis employing a single capillary column.

[0086]FIG. 3 is a side sectional view schematically showing oneembodiment applying the second aspect of the present invention to amulti-capillary electrophoretic apparatus, and its multi-capillaryarray-electrophoresis part comprises a detection side holder fixing partfixing a detection side holder and a parallelism adjusting mechanismadjusting the parallelism between a detection part and a part to bedetected.

[0087] A capillary array 2 is formed by arranging a plurality ofcapillary columns charged with gels of separation media. One ends (lowerends) 2 a of the capillary columns defining a sample injection side aretwo-dimensionally arranged and fixed by a sample injection side holder 4to come into contact with a sample in a sample injection reservoir or abuffer solution in a lower reservoir for electrophoresis. The other ends2 b of the capillary columns forming the capillary array 2 define adetection side on which the capillary columns are aligned with eachother by a detection side holder 6, and comes into contact with an upperreservoir buffer solution. A part to be detected 2 c is provided on thedetection side (2 b side) of the capillary array on a position where thecapillary columns are aligned with each other and supported by thedetection side holder 6.

[0088]FIGS. 4, 5 and 6 are a schematic front elevational view, aschematic left side elevational view, and a schematic top plan viewshowing an exemplary capillary array mounted on this embodiment

[0089] In the sample injection side holder 4, a rubber plate 4 d ofsilicone rubber holding and fixing glass capillary columns in/to holesis held between resin holder plates 4 a and 4 b for two-dimensionallyarranging the capillary columns and integrated by fixed screws 4 c. Theholder plates 4 a and 4 b are provided with holes for receiving thecapillary columns on 16 by 24 portions in correspondence to thepositions of holes of a 384-hole microplate used for sampleintroduction. The diameters of the holes of the holder plates 4 a and 4b are set larger than the outer diameters of the capillary columns. Thecapillary columns passing through the holder plates 4 a and 4 b and therubber plate 4 d held therebetween are held in the holes of the rubberplate 4 d by elasticity of rubber, to be airtightly fixed to the holder4.

[0090] The detection side holder 6 holds the capillary columns closelyarranged on a plane by a holder plate 6 a from below and by a rubberplate 6 d of silicone rubber from above. In order to press and fix thecapillary columns against and to the holder plate 6 a with the rubberplate 6 d, holder plate 6 b is provided for fixing the rubber plate 6 dto the holder plate 6 a on both side portions of the arrangement of thecapillary columns. Fixed screws 6 c fix the holder plates 6 a and 6 b toeach other.

[0091] The total length of each capillary column is about 500 nm, andthe part to be detected 2 c is provided on a position of about 400 nmfrom the end of the sample injection side. In order to form a detectionwindow on the part to be detected 2 c, the holder plates 6 a and 6 b andthe rubber plate 6 d are provided with elliptic openings 8 extending inthe direction of the arrangement of the capillary columns so that theopenings 8 overlap with each other on the part to be detected 2 c.Signal detection in electrophoresis is performed through the openings 8.

[0092] The multi-capillary electrophoretic apparatus according to thepresent invention is provided on the detection part with location pins44 a guiding the holder 6 to a fixed position as described later, andthe holder 6 is provided with location holes 44 b receiving the locationpins 44 a.

[0093] Each capillary column is made of quartz glass or borosilicateglass, and has an outer diameter of 200 to 300 μm and an inner diameterof 75 to 100 μm. The outer periphery of the capillary column ispreferably covered with a film of a non-fluorescent material such asSiO₂ not fluorescing or fluorescing to an extent not hinderingfluorescence measurement with excitation light of ultraviolet to nearinfrared regions. In this case, the film may not be removed on the partto be detected 2 c. If the capillary column has a fluorescing resinfilm, the film is removed on the part to be detected 2 c.

[0094] The capillary columns are charged with a polyacrylamide gel, alinear acrylamide gel, a polyethylene oxide (PEO) gel and the like asgels of separation media. Samples containing four types of DNA fragmentslabeled with four types of fluorescent materials selected from FAM, JOE,TAMRA, ROX, R6G, R-110 and the like varied with the end bases areinjected into the capillary columns respectively and simultaneouslyelectrophoresed.

[0095] Referring again to FIG. 3, an argon gas laser unit 10 is providedas an excitation light source for exciting the labeling fluorescentmaterials. The argon gas laser unit 10 is a multi-line type unit havingan output of 40 to 100 mW and simultaneously oscillates laser beamshaving wavelengths of 488 nm, 514.5 nm and the like.

[0096] When applying the multi-capillary electrophoretic apparatus shownin FIG. 3 to an apparatus utilizing multiphoton absorption of the firstaspect, a mode-locked titanium sapphire laser unit generating a laserbeam having a longer wavelength than fluorescence generated by a labeledfluorochrome is used as the excitation light source in place of theargon gas laser unit 10. The energy of one photon of the laser beam issmaller excitation energy for the fluorochrome.

[0097] An optical system 12 applying the laser beam from the laser unit10 to the part to be detected 2 c of the capillary array 2 as excitationlight and detecting fluorescence from the part to be detected 2 c is anepi-optical system shown in FIG. 7A in detail. Numeral 16 denotes amirror perpendicularly applying a laser beam 14 from the laser unit 10to a surface of the part to be detected 2 c of the capillary array 2,numeral 18 denotes a tunnel mirror having a hole on its center fortransmitting the excitation light beam through the hole and reflectingthe fluorescence on a mirror surface, and numeral 20 denotes anobjective lens consisting of a condenser lens condensing and projectingthe excitation light onto a single capillary column and receivingfluorescence generated from a sample migrating in the capillary column.The objective lens 20 projects the excitation light and receives thefluorescence by the same lens, and forms the epi-optical system. Themirror surface of the tunnel mirror 18 reflects the fluorescencecollected by the objective lens 20.

[0098] Numeral 22 denotes an optical filter blocking an excitation lightcomponent from the reflected light and transmitting the fluorescence,numeral 24 denotes a pinhole slit for limiting a detection field, andnumeral 26 denotes a diaphragm lens imaging the fluorescence transmittedthrough the optical filter 22 on the position of the pinhole slit 24. Afluorescing point in the capillary column is imaged on the position ofthe pinhole slit 24, thereby forming a confocal optical system. An edgefilter or colored glass can be employed as the optical filter 22 forremoving the excitation light. The pinhole slit 24 reduces the detectionfield for preventing invasion of stray light from adjacent capillarycolumns.

[0099] n order to divide the fluorescent image on the pinhole slit 24into four luminous fluxes, a lens panel 28 shown in FIG. 7B is arranged.The lens panel 28 can be manufactured as that prepared by cutting singlelenses and sticking the same to each other or a glass molding. A filterpanel 30 formed by different spectroscopic filters for respectivelabeling fluorescent materials shown in FIG. 7C is arranged on opticalpaths of the four luminous fluxes. The filter panel 30 is a bandpassfilter, which is formed by arranging four types of filters havingdifferent wavelength characteristics corresponding to the labelingfluorescent materials on the respective optical paths in parallel witheach other. The transmission wavelengths of the respective filterscorrespond to light emission wavelengths of the fluorescent materialslabeling fragment samples whose end bases are A (adenine), G (guanine),C (cytosine) and T (thymine). Four photomultiplier tubes 32 are arrangedon the respective optical paths for detecting fluorescence transmittedthrough the filters.

[0100] he epi-optical system 12 including the mirror 16, the tunnelmirror 18, the objective lens 20, the optical filter 22, the pinholeslit 24, the diaphragm lens 26, the lens panel 28, the filter panel 30and the photomultiplier tubes 32 is mounted on a stage of a scanningmechanism 34, and reciprocally moved along a straight line(perpendicular to the plane in FIG. 3 and vertical in FIG. 7A) parallelto the plane of the part to be detected 2 c of the capillary array 2 andperpendicular to the electrophoresis direction, in order to detectfluorescence from all capillary columns on the part to be detected 2 c.The laser beam 14 is incident upon the mirror 16 in parallel with ascanning direction of the epi-optical system 12, so that the opticalaxis of the laser beam 14 is not fluctuated by scanning of theepi-optical system 12.

[0101]FIG. 8A is a sectional view of one embodiment of the detectionside holder fixing part and its periphery as viewed from above, FIG. 8Bis a front sectional view taken along the line B-B′ in FIG. 8A and FIG.8C is a side sectional view taken along the line C-C′ in FIG. 8A, whileFIG. 8A is taken along the lines A-A′ in FIGS. 8B and 8C. FIGS. 8A and8B omit illustration of an upper electrode 58 and a detection sidecapillary end pressing member 60, and FIG. 8A also omits illustration ofa detected part pressing member 62.

[0102] A movable plate 36 is provided on a position for fixing thedetection side holder 6. FIGS. 9A, 9B and 9C are a top plan view, afront sectional view and a right side elevational view showing themovable plate 36 respectively.

[0103] The movable plate 36 is formed by a substrate 36 a and adetection position plate 36 b. The substrate 36 a is provided with aslot 38 in a direction where the epi-optical system 12 is scanned. Thedetection position plate 36 b slightly smaller in dimension than theopening 8 of the detection side holder 6 is arranged on the slot 38. Anepi-optical system scanning groove 40 is formed in the detectionposition plate 36 b on the substrate 36 a side, and a slot defining alight application window 42 is formed on the bottom surface of thescanning groove 40. The objective lens 20 side of the epi-optical system12 is arranged in the slot 38 and the scanning groove 40 and scannedalong the slot 38.

[0104] Two location pins 44 a are arranged on positions of the substrate36 a corresponding to the location holes 44 b of the holder 6. Theholder 6 is correctly arranged on the movable plate 36 by registeringthe positions of the location pins 44 a and the location holes 44 b andthose of the detection position plate 36 b and the opening 8 of theholder 6.

[0105] Furthermore, set screws 46 are arranged on the substrate 36 a onpositions corresponding to four comers of the holder 6. The height forarranging the holder 6 can be adjusted by rotating the set screws 46 andadjusting the length of the set screws 46 projecting from the substrate36 a.

[0106] Furthermore, the substrate 36 a is provided with a gate angleadjusting mechanism formed by two gate adjusting screws 48 a passingthrough the substrate 36 a and a gate adjusting supporting point pin 48b provided on a side opposed to the side provided with the gateadjusting screws 48 a and opposite to the detection position plate 36 b.The parallelism between the movable plate 36 and a scanning axis of theepi-optical system 12 can be adjusted by rotating the gate adjustingscrews 48 a.

[0107] The holder 6 is fixed to the movable plate 36 by fastening twoclamps 50 provided in the vicinity of both ends of the movable plate 36.

[0108] An upper reservoir 52 storing a buffer solution for dipping theother ends 2 b of the capillary columns forming the capillary array 2and a cover 54 covering upper portions of the movable plate 36 and theupper reservoir 52 are provided in the vicinity of the movable plate 36.The cover 54 can be opened/closed along a cover switching shaft 56.

[0109] The upper electrode 58 covered with a cylindrical insulatingmember is mounted on the cover 54 and comes into contact with the buffersolution of the upper reservoir 52 in the state covered with the cover54. A capillary array end pressing member 60 is arranged on the cover54, for bending the other ends 2 b of the capillary columns forming thecapillary array 2 toward the upper reservoir 52 and dipping the same inthe buffer solution.

[0110] The cover 54 is provided with the detected part pressing member62 on a position corresponding to the part to be detected 2 c. Thepressing member 62 is formed with a plane smaller in dimension than theopening 8 of the holder 6, and a rubber plate 64 of silicone rubber isstuck to this plane. Four rod members 66 provided on the cover 54 mountthe pressing member 62 to be slidable in a direction perpendicular tothe plane of the part to be detected 2 c. Springs 68 are arranged on therod members 66 between the cover 54 and the pressing member 62respectively, so that the pressing member 62 presses the part to bedetected 2 c against the detection position plate 36 b with appropriatepressure through the silicone rubber plate 64 when the cover 54 isclosed after arranging the holder 6 on the movable plate 36.

[0111] The detection side holder fixing part according to the presentinvention is formed by the movable plate 36, the gate adjusting screws48 a, the gate adjusting supporting point pin 48 b, the clamps 50 andthe detected part pressing member 62.

[0112] As shown in FIG. 3, the detection side holder 6 is fixed in anelectrophoresis chamber 66. A lower electrode 68 is mounted on a lowerportion of the chamber 66, to come into contact with a buffer solutionin a lower reservoir and communicate with the lower ends 2 a of thecapillary columns forming the capillary array 2 when the buffer solutionin a sample injection reservoir or the lower reservoir forelectrophoresis is pushed up to a position coming into contact with thelower ends 2 a of the capillary columns. A sample injection voltage oran electrophoresis voltage is applied between the buffer solutions inboth reservoirs from a high-voltage power source through the electrodes58 and 68. For example, the power supply voltage is 30 kV and a currentcapacity is 10 to 30 mA.

[0113] The reservoir for electrophoresis and a sample injectionreservoir 70 are arranged in a horizontal plane and supported on an X-Zsample stage 72 under the ends 2 a of the capillary columns on thesample injection side of the capillary array 2. The X-Z sample stage 72performs movement in a horizontal direction (X direction: perpendicularto the plane of FIG. 3) for locating either reservoir under the ends 2 aand movement in a vertical direction (Z direction: vertical in FIG. 3)for bringing the buffer solution in-the reservoir into contact with theends 2 a or separating the former from the latter by a sample stagemoving mechanism 74.

[0114] A sample titer plate 76 formed with wells corresponding to thearrangement of the ends 2 a of the capillary columns is placed on thereservoir 70. Bottoms of the wells pass through the sample titer plate76, membranes are formed on the bottoms and samples are adsorbed on themembranes of the wells. The buffer solution in the reservoir 70 comesinto contact with the membranes, and the sample injection voltage isapplied to the ends 2 a of the capillary columns from the lowerelectrode 68 through the buffer solution.

[0115] Operations of fixing the detection side holder 6 to the movableplate 36 and adjusting the parallelism between the plane of the part tobe detected 2 c and the scanning axis of the epi-optical system 12 shallnow be described.

[0116] The capillary array 2 having the part to be detected 2 c chargedwith a fluorochrome is prepared so that the holder 6 for the capillaryarray 2 is arranged on the movable plate 36 by opening the cover 54 andregistering the positions of the location pins 44 a and the locationholes 44 b and clamped and fixed by the clamps 50. Thus, the part to bedetected 2 c of the capillary array 2 can be fixed to the apparatus withexcellent reproducibility.

[0117] The cover 54 is closed so that the pressing member 62 presses thepart to be detected 2 c against the detection position plate 36 b andfixes the same onto the light application window 42 along the plane ofthe detection position plate 36 b while the pressing member 60 dips theends 2 b of the capillary columns of the capillary array 2 in the buffersolution of the upper reservoir 52.

[0118] The epi-optical system 12 is scanned and an image formed on thepinhole slit 24 is observed to determine whether or not the distancebetween the part to be detected 2 c and the objective lens 20 is proper.If the distance between the part to be detected 2 c and the objectivelens 20 is improper, the gate adjusting screws 48 a are rotated foradjusting the distance between the part to be detected 2 c and theobjective lens 20. Thus, requirement for working accuracy in preparationof the detection side holder 6 and formation of the capillary array 2can be relieved and reduction of detection sensitivity can besuppressed. Furthermore, since the distance between the part to bedetected 2 c and the epi-optical system 12 can be adjusted, thiselectrophoretic apparatus is adaptive to various outer diameters of thecapillary columns arranged on the capillary array 2. In addition, outerdiameter tolerance by manufacturing lot difference of the capillarycolumns can be allowed.

[0119] While this embodiment employs two gate adjusting screws and thegate adjusting supporting point pin as the gate angle adjustingmechanism, the present invention is not restricted to this but a gateangle may be automatically adjusted in correspondence to a detectionsignal at the time of scanning the epi-optical system by employing anactuator such as a piezoelectric element, for example.

[0120] Alteratively, an actuator moving the epi-optical system along theoptical axis of applied light may be provided in place of the gate angleadjusting mechanism for automatically adjusting the distance between thepart to be detected and the epi-optical system.

[0121]FIG. 10 is a perspective view of a capillary cassette of oneembodiment arranging a plurality of capillary columns.

[0122] A plurality of capillary columns 102 of a capillary array 2 arearranged, sample injection sides are fixed by a cassette holder 4 anddetection sides are fixed by a cassette holder 6 and a heat-shrinkabletube 80 on an end to form a capillary cassette 9. One ends 2 a of thecapillary columns 102 define a sample injection part and aretwo-dimensionally arranged and fixed by the cassette holder 4. The otherends 2 b of the capillary columns 102 forming the capillary cassette 9are planarly aligned with each other, fixed by the cassette holder 6,and cylindrically bundled by a shrank heat-shrinkable tube 80. Adetection window is formed on the cassette holder 6, and portions of thecapillary columns 2 located on the detection window define a part to bedetected 2 c.

[0123] For example, the outer diameter of each capillary column 102 is300 to 400 μm.

[0124]FIGS. 11A to 11C are model diagrams showing a procedure ofbundling the ends 2 b of the capillary columns 102 by theheat-shrinkable tube 80. FIGS. 12A and 12B are sectional views takenalong the lines A-A′ and B-B′ in FIG. 11C. These figures show thecapillary columns 102 in a reduced number.

[0125] Manufacturing is performed along the following sequence:

[0126] (A) After aligning the plurality of capillary columns 102 witheach other, a filler 12 such as an epoxy resin adhesive or a siliconcompound is applied to surface portions of the capillary columns 102 onprescribed positions from the ends 2 b.

[0127] (B) The ends 2 b of the capillary columns 102 are bundled andinserted into the heat-shrinkable tube 80, which in turn covers thepositions to which the filler 12 is applied.

[0128] (C) The heat-shrinkable tube 80 is heated and shrunk. The innerdiameter of the heat-shrinkable tube 80 is reduced, the capillarycolumns 102 adhere to each other, and clearances therebetween are filledup with the filler 12 so that the capillary columns 102 arecylindrically bundled in an airtight manner. Clearances between theheat-shrinkable tube 80 and the capillary columns 102 and between thecapillary columns 102 are sealed with the filler 12, and both ends ofthe heat-shrinkable tube 80 do not communicate with each other except inthe capillary columns 102.

[0129]FIG. 13 is a model diagram showing a method of fixing a mountingmember to a polymer charger to the ends 2 b of the capillary columns102.

[0130] The capillary columns 102 bundled by the heat-shrinkable tube 80are inserted into a ferrule 86 serving as the mounting member for thepolymer charger along with the heat-shrinkable tube 80 and fixed by ascrew 88 so that each capillary column 102 attains sufficient pressureresistance in polymer injection.

[0131] It is preferable to set the outer diameter of the shrankheat-shrinkable tube 80 receiving the capillary columns 102 to a pipeouter diameter of a liquid chromatograph such as φ1.6 (outer diameter of1.6 mm), φ2or φ3 by adjusting the outer diameters of the capillarycolumns 102, the number of the capillary columns 102 and the thicknessof the shrank heat-shrinkable tube 80. Consequently, an existing ferruleemployed for a liquid chromatograph or the like can be employed. Whenpreparing a dedicated ferrule, the capillary columns 102 may be bundledin response to the inner diameter of the dedicated ferrule.

[0132] Irregularities of the bundled capillary columns 102 are removeddue to the thickness of the heat-shrinkable tube 8, and hence thethickness of the shrunk heat-shrinkable tube 80 is preferably increased.

[0133] In polymer charging, the ferrule 86 is connected to and mountedon a connection part of the polymer charger, for charging the capillarycolumns 2 with polymers by press-filling or suction.

[0134]FIG. 14 is a schematic perspective view showing one embodiment ofa multi-capillary electrophoretic apparatus to which the capillarycassette 9 shown in FIG. 10 is applied. FIG. 14 omits illustration ofthe cassette holders 4 and 6. This multi-capillary electrophoreticapparatus is identical to that shown in FIG. 1 except the structure onthe side of the other ends 2 b, and hence redundant description isomitted.

[0135] The ends 2 b of the capillary columns are bundled by theheat-shrinkable tube 80, and dipped in a buffer solution 122 in areservoir 120 along with the heat-shrinkable tube 80.

[0136] Operations in electrophoretic separation are also identical tothose in FIG. 1.

[0137] Such a multi-capillary electrophoretic apparatus preferablycomprises an automatic polymer charging mechanism for reusing thecapillary columns 102 by discharging used polymers from the capillarycolumns 102 and charging new polymers.

[0138] Although the present invention has been described and illustratedin detail, it is clearly understood that the same is by way ofillustration and example only and is not to be taken by way oflimitation as the spirit and scope of the present invention are limitedonly by the terms of the appended claims.

What is claimed is:
 1. A capillary electrophoretic apparatus comprising;a capillary electrophoretic part for injecting a sample labeled with afluorochrome into an end of a capillary column and electrophoresing saidsample; and detection means applying excitation light having awavelength longer than the fluorescent wavelength of said fluorochrometo said fluorochrome and exciting the same by multiphoton absorption anddetecting fluorescence generated from said fluorochrome for detectingeach component separated in said capillary column on a appropriateposition of said capillary column.
 2. The capillary electrophoreticapparatus in accordance with claim 1, wherein said detection meanscomprises a laser unit having an oscillation wavelength in a range from400 nm to 2 μm as a light source.
 3. The capillary electrophoreticapparatus in accordance with claim 2, wherein said oscillationwavelength of said laser unit is at 700 to 900 nm.
 4. A multi-capillaryelectrophoretic apparatus comprising: a capillary array in which aplurality of capillary columns are arranged, one ends of said capillarycolumns defining sample injection sides are fixed by a sample injectionside holder, the other ends defining detection sides are aligned witheach other on a plane and fixed by a detection side holder and a part tobe detected is provided on positions of said detection sides of saidcapillary columns; a multi-capillary array electrophoresis part to whichsaid sample injection side holder and said detection side holder arefixed so that samples are injected into said capillary columns, saidsample injection side ends are dipped in a buffer solution, saiddetection side ends are dipped in another buffer solution and aelectrophoresis voltage is applied through both said buffer solutionsfor performing electrophoresis in all said capillary columns, saidmulti-capillary array electrophoresis part including a detection sideholder fixing part fixing said detection side holder and a parallelismadjusting mechanism adjusting the parallelism between a detection partand said part to be detected; and said detection part applying light tosaid part to be detected of said capillary array and detecting detectionlight by interaction between said light and said samples.
 5. Themulti-capillary electrophoretic apparatus in accordance with claim 4,wherein said detection part includes an epi-optical system condensingand projecting light onto one said capillary column on said part to bedetected while receiving light affected by interaction with said samplesand a scanning mechanism reciprocally moving said epi-optical systemalong a straight line parallel to the plane of arrangement on said partto be detected of said capillary array and perpendicular to theelectrophoresis direction, and said parallelism adjusting mechanismadjusts the parallelism between a scanning axis of said epi-opticalsystem and said part to be detected.
 6. The multi-capillaryelectrophoretic apparatus in accordance with claim 4, wherein saidparallelism adjusting mechanism is a gate adjusting mechanism adjustinga mounting angle of said detection side holder fixing part by rotationof a screw.
 7. The multi-capillary electrophoretic apparatus inaccordance with claim 5, wherein said parallelism adjusting mechanismincludes an actuator automatically adjusting a gate angle of saiddetection side holder fixing part in correspondence to a detectionsignal at the time when scanning said epi-optical system.
 8. Themulti-capillary electrophoretic apparatus in accordance with claim 5,wherein said detection side holder fixing part includes a detectionposition member arranged between said part to be detected and saidepi-optical system, formed with an opening on a position correspondingto said part to be detected and having a plane coming into contact withone surface of said part to be detected and a detected part pressingmember having a plane pressing said part to be detected against saiddetection position member from a side opposite to said detectionposition member.
 9. A multi-capillary electrophoretic apparatuscomprising: a capillary array in which a plurality of capillary columnsare arranged, one ends of said capillary columns defining sampleinjection sides are fixed by a sample injection side holder, the otherends defining detection sides are aligned with each other on a plane andfixed by a detection side holder and a part to be detected is providedon said detection side positions of said capillary columns; amulti-capillary array electrophoresis part to which said sampleinjection side holder and said detection side holder are fixed so thatsamples are injected into said capillary columns, said sample injectionside ends are dipped in a buffer solution, said detection side ends aredipped in another buffer solution and a electrophoresis voltage isapplied through both said buffer solutions for performingelectrophoresis in all said capillary columns; and an epi-optical systemcondensing and projecting light onto one said capillary column on saidpart to be detected of said capillary array and receiving light affectedby interaction with said samples, and a scanning mechanism reciprocallymoving said epi-optical system along a straight line perpendicular tothe electrophoresis direction while automatically adjusting the distancebetween said part to be detected and said epi-optical system incorrespondence to a detection signal at the time of performing scanningalong said straight line.
 10. A capillary cassette in which a pluralityof capillary columns used in a multi-capillary electrophoretic apparatusare bundled, wherein one ends thereof are cylindrically bundled by asleeve, and clearances between said sleeve and said capillary columnsand between said capillary columns are sealed with a filler.
 11. Thecapillary cassette in accordance with claim 10, wherein said sleeve isprepared by shrinking a shrinkable member.
 12. The capillary cassette inaccordance with claim 11, wherein said shrinkable member is aheat-shrinkable tube.
 13. The capillary cassette in accordance withclaim 10, wherein a mounting member for mounting on a gel charger whilekeeping air tightness between the same and said sleeve is mounted onsaid sleeve.
 14. The capillary cassette in accordance with claim 10,wherein a part to be detected on which said capillary columns arealigned with each other is provided on the side of said cylindricallybundled ends while said capillary columns are two-dimensionally arrangedon other ends to define a sample injection part.